A biosensor is an analytical device which converts a biological response into an electrical signal. Biosensors are used to detect a wide array of chemicals ranging from toxic agents and environmental pollutants to blood sugars. For example, biosensors have used surface enzyme reactions to detect low concentrations of biological substances and gases. The term ‘biosensor’ is often used to cover sensor devices used to determine the concentration of substances and other parameters of biological interest even where they do not directly utilize a biological system. While enzymes have been used as the biologically responsive material, other biological systems have been utilized as biosensors as well, such as, whole cell metabolism, ligand binding and the antibody-antigen reaction. A biosensor's composition will directly affect its shelf-life, response time, reproducibility, sensitivity, selectivity, and overall effectiveness.
Traditionally, films like cellulose acetate or nafion have been used as coatings to bind, or carry, enzymes in biosensors. The enzymes contained in the membrane or the coating of a biosensor detect analytes in the form of gases or other biological substances. These gases and biological substances act as substrates that attach to receptors located on the enzymes on the surface of a biosensor. Biosensors with higher surface areas allow larger quantities of enzymes to bind to their surface. Increased enzyme levels maximize a biosensor's ability to attract substrates and detect the presence of targeted substances. Nano-structured materials, including nanofibers, are miniscule-sized agents possessing high surface-area-to-mass ratios, rendering them extremely effective biosensors due to their ability to facilitate maximal enzyme loading on their surface.
Nano-structured materials for enzyme immobilization utilize the performance and flexibility of nanofibers in processing and use. These materials are particularly appealing whenever high enzyme loading or large amounts of surface area are of consideration. One method suitable for the preparation of nanofibers is electrospinning. Other methods include, for example, melt blowing of fibers or spinning split bicomponent fibers, among others.
Conventional enzymatic biosensors, however, suffer from a number of drawbacks affecting the mass transfer capabilities of the nano-structured system. Of these, four major drawbacks include low sensitivity, low stability, unreliability at low concentrations of analyte, and enzyme loss due to leaching-out in aqueous environments. In the latter instance, because native enzymes are generally considered water-soluble, extremely high loading is necessary to achieve a structure that retains suitable detection capabilities after the leaching-out of the enzyme.
There is, therefore, a need in the art for a novel method of electrospinning polymer-enzyme solution to prepare nanofibers with high enzyme loading, without serious mass transfer limitations. To address this need, the current invention provides a method of electrospinning polymer-enzyme solution, in an organic, non-aqueous solvent, which can be used to achieve high enzyme loading on polymeric nanofibers. Specifically, the system provided employs hydrophobic, solvent-based polymer electrospinning solution loaded with high amounts, up to 30% w/w of the polymer, organic-soluble enzymes. The use of this system avoids leaching problems experienced by water-soluble enzyme-containing systems, thus rendering the use of the electrospun fibers as biosensors much more feasible. In one embodiment, glucose oxidase is loaded on polyurethane nanofibers for use as a biosensor material.